The main aims of the research were to produce efficient nanofibrous filters with long-term antibacterial properties and to confirm the functionality of samples under real filtration conditions. A polyurethane solution was modified by micro- or nanoparticles of copper oxide in order to juxtapose the aggregation tendency of particles depending on their size. Modified solutions were electrospun by the Nanospider technique. The roller spinning electrode with a needle surface and static wire electrode were used for the production of functionalized nanofibers. The antibacterial properties of the modified nanofibrous layers were studied under simulated conditions of water and air filtration. Particular attention was paid to the fixation mechanism of modifiers in the structure of filters. It was determined that the rotating electrode with the needle surface is more efficient for the spinning of composite solutions due to the continuous mixing and the avoidance of particle precipitation at the bottom of the bath with modified polyurethane. Moreover, it was possible to state that microparticles of copper oxide are more appropriate antimicrobial additives due to their weaker aggregation tendency but stronger fixation in the fibrous structure than nanoparticles. From the results, it is possible to conclude that nanofibers with well-studied durable antibacterial properties may be recommended as excellent materials for water and air filtration applications.